Method and peptide for regulating cellular activity

ABSTRACT

Method and peptide for regulating cellular activity includes a panel of synthesized peptides that have biological effects on inhibiting or enhancing cellular activity. Selected peptides can be used as therapy to reduce and/or inhibit, or initiate and/or enhance, an inflammatory response in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/656,512,filed Jan. 23, 2007, which is a divisional of U.S. patent applicationSer. No. 11/178,316, filed Jul. 12, 2005, which claims priority on priorU.S. Provisional Application No. 60/586,701, filed Jul. 12, 2004. Theprior applications are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work leading to the present invention was supported by one or moregrants from the U.S. Government, including Veterans Affairs RR&D GrantC2870R(SHH), NIH Grant (NIDCD R01 DC05593 (DRT and SHH), and SBIR IR43DC05882-01 (SEK)). The U.S. Government therefore has certain rights inthe invention.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference the Sequence Listing,including SEQ ID NO: 1 to SEQ ID NO: 21 provided herewith.

FIELD AND HISTORICAL BACKGROUND OF THE INVENTION

The present invention is generally directed to a method and peptide forregulating cellular activity, and more particularly to theidentification and use of various peptides derived from vaccinia virusA52R protein that have biological effects by inhibiting or enhancingcellular activity, and particularly toll-like receptors (TLR) inducedcytokine secretion in a cell.

The innate immune system, involved in both the detection and control ofinfection, recognizes conserved motifs from pathogens termedpathogen-associated molecular patterns (PAMPs) (References 1 and 2).Toll-like receptors (TLRs) recognize PAMPs, and their interactiontriggers a series of intracellular signaling events that culminates inexpression of cell-surface molecules, secretion of pro-inflammatorycytokines and induction of acquired immunity (References 2-9). TLRs arecharacterized by an extracellular leucine-rich repeat motif and anintracellular Toll/IL-1 receptor (TIR) domain. Pathogens are detected bythe innate immune system, with recognition of the bacterial PAMPs LPS,CpG DNA, flagellin, and peptidoglycan mediated by TLR4, TLR9, TLR5, andTLR2 respectively (References 4 and 10-13). Recognition of viralinfections are mediated primarily by TLR3 in response to viral dsRNA(Reference 14). Cell activation in response to different PAMPs involvesa number of intracellular molecules common to all TLRs, including MyD88,members of the IL-1 receptor-associated kinase (IRAK) proteins, TNFreceptor associated factor (TRAF6), and NF-κB (Reference 1).

Vaccinia virus, a member of the poxvirus family, is a DNA virus that hasbeen demonstrated to encode immunomodulatory proteins (References15-18). One of these proteins, A52R, has been shown to inhibit NF-κBactivation following initiation of the TIR signaling cascade (References15 and 18). Recent studies have demonstrated that A52R inhibits TIRsignaling and contributes to the virulence of vaccinia virus. Inhibitionof TIR signaling by A52R is mediated by binding of the protein to bothTRAF6 and IRAK2 (Reference 18).

The present invention is directed to the identification andcharacterization of a peptide, derived from the A52R protein, thatsignificantly inhibits in vitro cytokine production in response to bothbacterial and viral PAMPs. This peptide has characteristics consistentwith a reagent that inhibits intracellular signaling triggered by TLRactivation. Cytokine secretion induced by non-TLR stimulation was notinhibited by the peptide. The in vivo activity of this peptide wasdemonstrated by dramatically reducing middle ear inflammation in miceinjected with heat-inactivated Streptococcus pneumoniae (S. pneumoniae).This peptide may have application in the treatment of this and otherinflammatory conditions that result from ongoing TLR activation. Inaddition, we have also identified three distinct A52R peptides thatinhibit cytokine secretion and five other distinct peptides that enhancecytokine secretion.

OBJECTS AND SUMMARY OF THE INVENTION

The main object of the present invention is to provide a method andpeptide for regulating cellular activity.

Another object of the present invention is to provide a peptide forinhibiting cellular activity.

Another object of the present invention is to provide a peptide forenhancing cellular activity.

Another object of the present invention is to provide a peptide that canbe used to reduce and/or inhibit pathogen associated inflammation.

Another object of the present invention is to provide a peptide that canbe used to reduce and/or inhibit self-antigen associated inflammation.

Another object of the present invention is to provide a peptide that canbe used to reduce and/or inhibit antigen associated inflammation.

Another object of the present invention is to provide a peptide that canbe used to initiate and/or enhance pathogen associated inflammation.

Another object of the present invention is to provide a peptide that canbe used to initiate and/or enhance self-antigen associated inflammation.

Another object of the present invention is to provide a peptide that canbe used to initiate and/or enhance antigen associated inflammation.

Another object of the present invention is to provide a peptide thatinhibits cytokine secretion in response to TLR activation.

Another object of the present invention is to provide a peptide thatinhibits cytokine secretion by interaction with an intracellular portionof the TIR pathway upstream of IκB.

Another object of the present invention is to provide a peptide thatinhibits cytokine secretion by interaction in the TIR/TLR signalingpathway.

Another object of the present invention is to provide a peptide thatinhibits in vitro cytokine production in response to bacterial and/orviral pathogen-associated molecular patterns (PAMPs).

Another object of the present invention is to provide a peptide thatreduces and/or inhibits in vivo inflammation, and particularly bacterialand/or viral-induced inflammation.

Another object of the present invention is to provide a peptide as setforth in SEQ ID NO: 1 to SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO:21.

Another object of the present invention is to provide a peptide thatreduces and/or inhibits fluid secretion and/or accumulation into themiddle ear of a subject.

Another object of the present invention is to provide a peptide thatreduces and/or inhibits mucosal cellular hypertrophy in the middle earof a subject.

Another object of the present invention is to provide a peptide that canbe used in diagnostic, therapeutic, and/or other applications.

In summary, toll-like receptors recognize and respond to conservedmotifs termed pathogen-associated molecular patterns (PAMPs). TLRs arecharacterized by an extracellular leucine-rich repeat motif and anintracellular Toll/IL-1 receptor (TIR) domain. Triggering of TLRs byPAMPs initiates a series of intracellular signaling events resulting inan inflammatory immune response designed to contain and eliminate thepathogen. Vaccinia virus encodes immunoregulatory proteins, such asA52R, that can effectively inhibit intracellular TIR signaling resultingin a diminished host immune response and enhancing viral survival. Thepresent invention is directed to the identification and characterizationof a peptide derived from the A52R protein (sequence DIVKLTVYDCI—SEQ IDNO: 13) that when linked to a 9-arginine cell transduction sequenceeffectively inhibits cytokine secretion in response to TLR activation.The peptide had no effect on cytokine secretion resulting from cellactivation that was initiated independent of TLR stimulation. Employinga mouse model of otitis media with effusion (OME), administration ofheat-inactivated Streptococcus pneumoniae (S. pneumoniae) into themiddle ears of BALB/c mice resulted in a significant inflammatoryresponse that was dramatically reduced with peptide treatment.Experiments have also demonstrated that the peptide will reducepro-inflammatory mediators in a mouse model of LPS-induced septic shock.The identification of this peptide that selectively targetsTLR-dependent signaling may have application in the treatment of chronicinflammation initiated by bacterial or viral infections. In addition tothe peptide described above, we have identified three additionaldistinct peptides from the A52R protein that also inhibit cytokinesecretion, and five other distinct peptides that demonstrated enhancedcytokine secretion.

At least one of the above objects is met, in part, by the presentinvention which in one aspect includes a method of regulating cellularactivity, including subjecting a cell to a peptide derived from thevaccinia virus A52R protein.

Another aspect of the present invention includes a method of inhibitingTLR-induced cytokine secretion in a cell, including subjecting a cell toa peptide derived from the vaccinia virus A52R protein.

Another aspect of the present invention includes a method of enhancingTLR-induced cytokine secretion in a cell, including subjecting a cell toa peptide derived from the vaccinia virus A52R protein.

Another aspect of the present invention includes a method of reducing orinhibiting inflammation in a subject, including administering aneffective amount of a peptide derived from the vaccinia virus A52Rprotein in a subject in need thereof.

Another aspect of the present invention includes a method of initiatingor enhancing an inflammatory response in a subject, includingadministering an effective amount of a peptide derived from the vacciniavirus A52R protein in a subject in need thereof.

Another aspect of the present invention includes a synthesized peptide,including at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 18, and a combination thereof.

Another aspect of the present invention includes an immunoregulatorypeptide, including at least one amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 18, and a combinationthereof.

Another aspect of the present invention includes a fusion peptide,including at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 18, and a combination thereof,and a marker protein and/or a peptide tag.

Another aspect of the present invention includes a complementary peptidefor interacting with at least one of the peptides set forth in SEQ IDNO: 1 to SEQ ID NO: 18, and a combination thereof.

Another aspect of the present invention includes an antibody specificfor binding to at least one of the peptides set forth in SEQ ID NO: 1 toSEQ ID NO: 18, and a combination thereof.

Another aspect of the present invention includes a pharmaceuticalcomposition including at least one of the peptides set forth in SEQ IDNO: 1 to SEQ ID NO: 18, and a combination thereof.

Another aspect of the present invention includes a synthesized peptideas set forth in SEQ ID NO: 20.

BRIEF DESCRIPTION OF THE DRAWINGS

One of the above and other objects, novel features and advantages of thepresent invention will become apparent from the following detaileddescription of the preferred embodiment(s) of the invention, asillustrated in the drawings, in which:

FIG. 1 illustrates internalization of peptide P13 requires the celltransduction sequence: RAW264.7 cells were incubated with 10 μM peptidecontaining the transducing sequence (DIVKLTVYDCI-RRRRRRRRR; solidline—SEQ ID NO: 20) or with 10 μM peptide lacking the transducingsequence (DIVKLTVYDCI; dashed line—SEQ ID NO: 13) for 15 minutes andinternalization of FITC-peptide evaluated by fluorescent-activated cellsorter (FACS);

FIG. 2 illustrates the effect of peptides of the present invention onMIP-2 secretion: RAW264.7 cells were incubated 15 minutes with eithermedium (no peptide) or individual peptides at the maximal concentrationsthat did not affect cell viability. The cells were then stimulated withCpG-ODN (1 μg/ml) for 18 hours, cell-free supernatants analyzed forMIP-2 by ELISA, and data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 3 illustrates inhibition of MIP-2 secretion by peptide P13:RAW264.7 cells were incubated 15 minutes with either media (no peptide),5 μM, 8 μM, or 10 μM peptide P13 and then stimulated with CpG-ODN (1μg/ml) for 18 hours. Cell-free supernatants were analyzed for MIP-2 byELISA and data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 4 illustrates inhibition of MIP-2 secretion by peptide P13:RAW264.7 cells were incubated 15 minutes with either media (no peptide),5 μM, 8 μM, or 10 μM peptide P13 and then stimulated with LPS (1 ng/ml)for 18 hours. Cell-free supernatants were analyzed for MIP-2 by ELISAand data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 5 illustrates inhibition of MIP-2 secretion by peptide P13:RAW264.7 cells were incubated 15 minutes with either media (no peptide),5 μM, 8 μM, or 10 μM peptide P13 and then stimulated with Poly(I:C) (10μg/ml) for 18 hours. Cell-free supernatants were analyzed for MIP-2 byELISA and data expressed as MIP-2 ng/ml +/−S.D.,

FIG. 6 illustrates inhibition of MIP-2 secretion by peptide P13:RAW264.7 cells were incubated 15 minutes with either media (no peptide),5 μM, 8 μM, or 10 μM peptide P13 and then stimulated with flagellin (5ng/ml) for 18 hours. Cell-free supernatants were analyzed for MIP-2 byELISA and data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 7 illustrates inhibition of MIP-2 secretion by peptide P13:RAW264.7 cells were incubated 15 minutes with either media (no peptide),5 μM, 8 μM, or 10 μM peptide P13 and then stimulated with zymosan (10μg/ml) for 18 hours. Cell-free supernatants were analyzed for MIP-2 byELISA and data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 8 illustrates that peptide P13 inhibits MIP-2 secretion fromactivated cells: RAW264.7 cells were incubated with peptide P13 forvarious times; either before (15 minutes), simultaneous with (time 0),or after (0.25, 0.5, 1, 2, 3, or 4 hrs) stimulation for 18 hours withCpG-ODN (1 μg/ml). Positive control was cells stimulated with CpG-ODN (1μg/ml) without added peptide. Cell-free supernatants were analyzed forMIP-2 by ELISA and data expressed as MIP-2 ng/ml +/−S.D.;

FIG. 9 illustrates peptide P13 inhibition of TNF-α secretion: RAW264.7cells were incubated for 15 minutes with 10 μM peptide P13 and thenstimulated with CpG-ODN (1 μg/ml). Cell-free supernatants were collectedafter 18 hours and cytokine secretion quantified by ELISA and dataexpressed as ng/ml +/−S.D.;

FIG. 10 illustrates peptide P13 inhibition of IL-6 secretion: RAW264.7cells were incubated for 15 minutes with 10 μM peptide P13 and thenstimulated with CpG-ODN (1 μg/ml). Cell-free supernatants were collectedafter 18 hours and cytokine secretion quantified by ELISA and dataexpressed as ng/ml +/−S.D.;

FIG. 11 illustrates peptide P13 inhibition of IL-10 secretion: RAW264.7cells were incubated for 15 minutes with 10 μM peptide P13 and thenstimulated with CpG-ODN (1 μg/ml). Cell-free supernatants were collectedafter 18 hours and cytokine secretion quantified by ELISA and dataexpressed as ng/ml +/−S.D.;

FIG. 12 is a photographic illustration of the middle ear from aftertreatment with peptide P13 The normal middle ear of BALB/c mice is clearbetween the tympanic membrane (TM) and round window (RW);

FIG. 13 is a photographic illustration of the mucosal epithelium aftertreatment with peptide P13. The normal mucosal epithelium (E) of themiddle ear lateral wall is typically comprised of only 1-2 layers of lowcuboidal cells;

FIG. 14 is a photographic illustration showing that injection of themiddle ear with Streptococcus pneumoniae (S. Pneu.) causes extensiveinflammation. The middle ear space lateral to the cochlea (C) and aroundthe stapedial artery (V) is filled with fluid and the tympanic membraneis thickened;

FIG. 15 is a higher magnification photographic illustration of the S.pneu injected mouse showing the extensive fluid accumulation in themiddle ear space;

FIG. 16 is a photographic illustration showing that S. pneumoniae (S.pneu.) injection causes hypertrophy of the epithelium, cells becomesecretory, and fluid accumulates;

FIG. 17 is a photographic illustration showing that the fluid andinflammation is significantly reduced and confined to the round windowarea when peptide P13 (S. pneu.+Pep) is injected with the bacteria;

FIG. 18 is a photographic illustration showing that the epitheliumretains a normal appearance and fluid disappears when peptide P13 isinjected along with S. pneumoniae (S. pneu.+Pep);

FIGS. 19A-B illustrate the amino acid sequences (without thenine-arginine cell transducing sequence) of various peptides constructedfrom the sequence of the vaccinia virus A52R, in accordance with thepresent invention;

FIG. 20 illustrates the effect of peptides on MIP-2 secretion: RAW264.7cells were incubated 15 minutes with either medium (no peptide) orindividual peptides at the maximal concentrations that did not affectcell viability. The cells were then stimulated with LPS (1 ng/ml) for 18hours, cell-free supernatants analyzed for MIP-2 by ELISA, and dataexpressed as MIP-2 ng/ml +/−S.D.;

FIG. 21 illustrates the effect of peptides on MIP-2 secretion: RAW264.7cells were incubated 15 minutes with either medium (no peptide) orindividual peptides at the maximal concentrations that did not affectcell viability. The cells were then stimulated with poly(I:C) (10 μg/ml)for 18 hours, cell-free supernatants analyzed for MIP-2 by ELISA, anddata expressed as MIP-2 ng/ml +/−S.D.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) OF THE INVENTION

TLRs are conserved molecular receptors that recognize structures frombacteria, fungi, protozoa, and viruses. Activation of TLRs initiates aseries of intracellular events resulting in an innate immune responsecharacterized by the production of pro-inflammatory cytokines(References 2-9). TLR signaling originates from the cytoplasmic TIRdomain, conserved among all TLRs. The adapter molecule MyD88, containingboth a TIR domain and a death domain, associates with the TIR domain ofTLRs and IRAK proteins. Phosporylation of IRAK leads to association withTRAF6 and subsequent activation of NF-κB and secretion ofpro-inflammatory cytokines (References 14, 22-25). A52R, animmunoregulatory protein from vaccinia virus, has previously been shownto be an intracellular inhibitor of TIR-dependent signaling (References15 and 18). When expressed in HEK293 cells, A52R was shown to inhibitNF-κB activation in response to stimulation by a variety of TLRs,including TLR4, TLR5, and the combination of TLR2 and 6, and TLR 2and 1. In addition, A52R inhibited NF-κB activation in response toPoly(I:C), a synthetic ligand for TLR3. TLR3 has been implicated in ananti-viral innate immune response. The peptide P13 (sequenceDIVKLTVYDCI—SEQ ID NO: 13) a subject of the present invention, wasderived from the immunoregulatory protein A52R and demonstrates many ofthe same properties as the protein. The peptide inhibits cytokinesecretion in response to a variety of TLR ligands, including LPS(lipopolysaccharide) (TLR4), CpG-ODN (TLR9), Poly(I:C) (TLR3), flaggelin(TLR5), and zymosan (TLR2). Harte and colleagues (Reference 18) havedemonstrated that the A52R protein inhibits TIR signaling by binding toboth IRAK2 and TRAF6, key intracellular regulatory proteins. Theseauthors further suggest that A52R binds independently to IRAK2 andTRAF6, suggesting the redundant targeting may indicate the importance ofinhibiting TIR activation to enhance virulence. Consistent with thisspeculation, deletion of the A52R protein from vaccinia virus resultedin reduced viral virulence. The mechanism by which peptide P13 inhibitsTIR-dependent cytokine secretion remains to be defined. Our studiesdemonstrated that internalization of the peptide was required forinhibition and that cytokine secretion, in response to non-TLR dependentactivation, was not affected. In addition, the demonstration thatpeptide P13 inhibited phosphorylation of IκB-α and inhibited TLR3signaling, is consistent with the hypothesis that P13 acts on the TIRsignaling pathway at some point between TRAF6 and IκB. Whether peptideP13 associates with TRAF6, like the parent A52R protein, or anotherintracellular signaling protein further downstream, is currently underinvestigation.

The in vivo effectiveness of the peptide was demonstrated using a mousemodel of OME. OME is an inflammatory disease of the middle earaccompanied by fluid accumulation. It is characterized by aninfiltration of leukocytes, macrophages and mast cells and release ofinflammatory mediators and enzymes (Reference 21). These mediatorsincrease vascular permeability and secretory activity, and initiate acascade of inflammatory events, resulting in fluid accumulation andmucin secretion (References 26 and 27). The initiation of inflammationin OME has been attributed to a variety of factors, including bacterialor viral infections, Eustachian tube dysfunction, and allergy. However,the evidence points to a bacterial etiology leading to cytokineactivation in the majority of cases. Bacteria have been cultured from upto 40% of effusions and studies have shown bacterial DNA by PCR inapproximately 80% of effusions, often in the absence of viable organismsin culture (Reference 28). The most common bacteria invading the middleear are S. pneumoniae, H. influenzae, and M. catarralis. These threebacteria account for 85% of acute middle ear infections (Reference 27),with S. pneumoniae being the most frequent cause. Initially, livebacteria trigger acute inflammation, which is designed to eliminate thepathogen. During acute infection, interference with the innate immuneresponse would be potentially harmful to the host and may lead tofurther bacterial spread. Acute inflammation initiated by bacterialinfections self-resolves or is treatable by antibiotics. Chronicinflammation involves continued activation of the immune system, oftenby non-viable bacterial products. OME is often prolonged or anti-bioticresistant, suggesting TLR stimulation in absence of live bacteria. Wewould predict that agents that interfere with TLR-dependent signalingwould be potential treatments for prolonged or antibiotic resistantmiddle ear inflammation. In our studies, treatment of mice with peptideP13 resulted in a significant reduction in bacterial-inducedinflammation in the middle ear. Fluid accumulation, infiltrating cells,and tympanic membrane thickness in the middle ear were all dramaticallyreduced with peptide treatment. Administration of heat-inactivatedbacteria, which has a number of potential TLR ligands, induced aninflammatory response in the middle ear most likely resulting fromactivation of multiple TLRs. In our studies, the use of heat-inactivatedbacteria allowed for an examination of peptide inhibition ofinflammation without the potential for bacterial spread that may occurin an acute infection initiated with live bacteria. The ability ofpeptide P13 to significantly inhibit this response in vivo is consistentwith the in vitro data showing inhibition of cytokine secretion inresponse to multiple TLR ligands used either individually or incombination. In these studies a single dose of peptide was administeredat the same time as heat-inactivated S. pneumoniae into the middle earsof normal BALB/c mice. While these studies demonstrated a dramaticeffect on inflammation, additional studies assessing the effect ofpeptide treatment on resolving an ongoing inflammatory response areneeded. Of interest in this respect, our in vitro data showed inhibitionof cytokine secretion even when peptide P13 was added several hoursafter initiation of TLR activation.

The initiation of an inflammatory response to pathogens is a criticalcomponent of the innate immune response and is designed to controlinfection. However, the sustained production of inflammatory mediatorscan lead to chronic inflammation, tissue damage and disease development.The signaling cascade initiated by PAMP/TLR interactions and culminatingin cell activation has been associated with many disease states,including sepsis, autoimmune diseases, asthma, heart disease and cancer(Reference 29). For example, it is hypothesized that sepsis occurs whenbacteria and their products activate an uncontrolled network ofhost-derived mediators, such as pro-inflammatory cytokines which canlead to multi-organ failure, cardiovascular collapse and death. Anabnormal TLR signaling response could lead to exaggeratedcell-activation responses contributing to sepsis (Reference 30 and 31).Inflammation is also a key aspect of autoimmunity, and is hypothesizedto play a role in tissue destruction in diseases such as multiplesclerosis, rheumatoid arthritis and insulin-dependent diabetes mellitus(Reference 32). Cells of the innate immune system have an essential rolein acquired/adaptive immunity. TLR proteins are involved in thematuration and activation of dendritic cells, the antigen-presentingcell type considered most relevant to development of acquired immunity(Reference 33). Allergic asthma is an example of a chronic inflammatorydisease with an adaptive immune response, and the TLR signaling pathwayis hypothesized to play an important role in the induction phase of anallergic phenotype (Reference 30). Bacterial and viral infections,causing increased inflammatory cell activation, are the main cause ofexacerbations in diseases such as asthma and COPD (chronic obstructivepulmonary disease) (Reference 30). Understanding and manipulating theTLR cell activation pathway has the potential to provide therapeuticbenefit for a variety of diseases with an inflammatory etiology.Treatments for inflammation have included the use of aspirin andglucocorticoids to block NF-κB activation (References 29, 34, 35) andthe targeting of specific inflammatory mediators such as TNF-α(Reference 36). Recent studies report blocking the interaction of TLRsand their ligands (Reference 37), or suppressing TLR expression(References 38-40) may provide new approaches for controllinginflammation. The identification of proteins involved in TIR signaling,and their molecular characterization, have lead to development of agentsto inhibit specific points within the TIR signaling cascade. Bartfai andcolleagues (Reference 41) have recently reported the synthesis of a lowmolecular weight mimic of MyD88. The structure of the compound was basedon the sequence of the TIR domain. The compound inhibited theinteraction between MyD88 and the IL-1R1 TIR domain, thereby inhibitingIL-1 induced activation in vitro and was effective in vivo at blockingIL-1 induced fever in mice. The compound did not block the interactionof TLR4 and MyD88 and therefore LPS induced activation was notinhibited. Inhibition of multiple TLR-dependent responses, by targetinga common signaling component, may prove to be a more effective approachto controlling an inflammatory response.

In the present invention, we identify an 11 amino acid sequence from thevaccinia virus A52R protein that has many of the same immunoregulatoryproperties described for the whole protein. When linked with a celltransducing sequence, our experiments showed this peptide inhibited invitro TLR-induced cytokine secretion and in vivo significantly reducedbacterial-induced inflammation in a murine model of OME. In addition, wehave identified a panel of other distinct A52R derived peptides thateither inhibit or enhance cytokine secretion in response toTLR-dependent stimulation. The treatment and control of bacterial andviral-induced inflammation represents a significant clinical challenge.The selective targeting of the TLR/TIR signaling cascade represents oneapproach to control inflammation and the identification of thesepeptides from the A52R protein may have potential therapeuticapplication.

The peptide 13 sequence was derived from the A52R sequence from vacciniavirus. Blast search analysis shows that peptide P13 has 100% homologywith two proteins from vaccinia virus other than A52R, two proteins fromcowpox virus, and one protein from rabbit pox virus. Peptide 13 wasshown to have significant homology with three separate proteins fromthree different strains of variola (smallpox) virus: i) A46L fromvariola major virus strain India ii) A49L from variola minor virusGarcia iii) A44L from variola major virus strain Bangladesh.

Materials and Methods

Peptide synthesis: Peptides were synthesized containing an 11-18 aminoacid sequence from the vaccinia virus A52R protein and a 9-residuearginine cell transduction sequence positioned at the COOH-terminal endto allow for cell internalization. Each peptide was constructed bothwith and without a FITC-label (Fluorescein isothiocyanate). FITC labeledpeptides were used for FACS analysis. The peptides lacking the FITClabel were used for in vitro inhibition assays and in vivo treatmentstudies.

Reagents: Nuclease-resistant phosphorylated oligonucleotide waspurchased from Oligos, Inc. (Wilsonville, Oreg.). The sequence was5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 19) (CpG ODN) (Reference 19).Mouse IL-1α and TNF-α were purchased from R&D Systems. PMA (phorbolmyristate acetate) and LPS (Lipopolysaccaride) were purchased fromSigma. The TLR ligands flagellin, zymosan, and Poly(I:C) were purchasedfrom Invivogen. Cytokine assays were performed using assay kitspurchased from R&D Systems. (Heat-inactivated S. pneumoniae was the kindgift of Dr. Thomas DeMaria, The Ohio State University College ofMedicine, Department of Otolaryngology, Columbus, Ohio.)

Cell lines and Cultures: RAW264.7 (murine monocyte/macrophage) cells(American Type Culture Collection) were cultured at 37° C. in a 5% CO₂humidified incubator and grown in DMEM (Dulbecco's Modified EagleMedium) (Gibco) supplemented with 10% (v/v) heat-inactivated FCS, 1.5 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Cytokine secretion: RAW264.7 cells were plated at 1.5×10⁵ cells/well in48 well plates. After 24 hours, the cells were incubated with peptide atvarious concentrations at room temperature in triplicate either before,simultaneous with, or after activation with various bacterial or viralPAMPs for 18 hours. Cell-free supernatants were analyzed for cytokinesby ELISA, in quadruplicate. RAW264.7 cells were stimulated with eitherCpG-ODN (1 μg/ml), LPS (1 ng/ml), Poly(I:C) (10 μg/ml), flagellin (5ng/ml), or zymosan (10 μg/ml). Dose response curves were done with eachPAMP to determine optimal stimulation concentration.

Flow Cytometry: Cells were analyzed by flow cytometry (FACScan, BectonDickinson) using Cellquest software to quantify internalization ofpeptides. Gates were drawn to exclude dead cells based on 7-AAD(7-amino-actinomycin D) staining. Flourescence due to cell-surfacebinding of FITC-labeled peptides was quenched using trypan blue. Dataobtained were geometric mean fluorescent units (F) with backgroundautofluorescence subtracted.

Immunoblotting: RAW264.7 cells (6×10⁵) were plated in 12 well platesovernight. Cells were incubated for 15 minutes at room temperature witheither peptide P13 or control scrambled peptide, and then stimulatedwith medium or LPS (1 ng/ml) for either 15 or 30 minutes. Cells werelysed, and proteins fractionated by SDS/PAGE (Sodium DodecylSulfate/Polyacrylamide Gel Electrophoresis) (12%). Immunoblotting wasdone using Phospho-IκB-α (Ser32) antibody (Cell Signaling), detectedusing horseradish peroxidase-conjugated secondary antibody, andvisualized by chemiluminescence. Measurements of band intensity weremade using the Nucleo Tech Gel Expert software linked to an Epsonexpression 636 scanner and expressed as intensity/area.

Induction of otitis media: BALB/c (Bagg albino) mice, 8-12 weeks of age,were anesthetized with a subcutaneous injection of xylazine & ketamine(0.1 mg/30 gm body weight) and their ears examined under the operatingmicroscope to assure they were free of infection or perforation. Onegroup of animals (n=5) was injected with PBS (Phospho-buffered saline)in one ear and with 10 μM peptide in the opposite ear, to determine theeffect of peptide without added bacteria. A second group of animals(n=20) received 5.0 t of PBS plus heat-inactivated S. pneumoniae (10⁹CFU/ml) in one ear and 5.0 μl of peptide (10 μM) plus heat-inactivatedS. pneumoniae (10⁹ CFU/ml) in the opposite ear. Injections were donethrough the tympanic membrane. Animals were killed 3 days afterbacterial injection and tissue histologically processed to assess middleear disease. Inflammation was quantified by measuring 1) area of fluidpresent in the middle ear, 2) number of cells in middle ear fluid, and3) thickness of the tympanic membrane (TM) taken at a point away fromthe injection site. From previous studies, data was obtained from mice(n=18) injected with PBS alone for each of the histological parametersmeasured, to serve as a control group. Disease induction was defined aspositive if the ear injected with S. pneumoniae without peptidedemonstrated an increase of at least two standard deviations above thecontrol PBS treated mice in at least two of the three parametersassessed for middle ear inflammation: Fluid area, cell number, thicknessof the tympanic membrane. Inflammation was successfully induced in 7 of20 mice.

Tissue Collection: At the end of the experimental treatment, mice werekilled and tissues collected for histology. Mice were overdosed onanesthetic and perfused intracardially with 1.0 ml of saline, followedby 20 ml of fixative (1.5% paraformaldehyde-3% glutaraldehyde in 0.1 Mphosphate buffer). The middle ears were left intact and connected toeach other by the skull base so both ears were processed together forhistology and sectioning. This enables all histologic embedding,sectioning, staining, and analysis to be done on the two sidessimultaneously to reduce any impact of processing variables on thesubsequent quantitative analyses. Middle ears were decalcified, embeddedin glycol methacrylate plastic, sectioned at 5 μm, mounted serially onglass slides, stained, and coverslipped.

Histopathologic Analysis: Three consecutive sections at the level of theumbo and promontory were selected for measures of 1) area of fluidpresent in the middle ear, 2) number of cells in middle ear fluid, and3) thickness of the tympanic membrane. Each measurement was taken on thethree sequential sections per specimen. The value presented for eachparameter represents the mean of the three sections.

Statistical analyses: To determine the effect of peptide without addedbacteria, animals (n=5) were injected in one ear with PBS alone, and theother ear with 10 μM peptide. Paired t-tests were done comparing theeffect of PBS alone with the effect of peptide for each of the 3histological parameters 1) area of fluid present in the middle ear, 2)number of cells in middle ear fluid, and 3) thickness of the tympanicmembrane. Out of 20 animals injected with S. pneumoniae, seven met thecriteria for disease induction. Paired t-tests were done using theseseven animals comparing the effect of peptide plus S. pneumoniae in oneear with S. pneumoniae alone in the opposite ear for each of thehistological parameters described above.

Results

Peptide construction: The A52R protein from vaccinia virus haspreviously been shown to inhibit intracellular TIR signaling (References15 and 18). To investigate which amino acid sequence(s) of A52R wasresponsible for this inhibitory effect, we constructed 18 peptides whosedesign was based on the sequence of the vaccinia virus A52R protein.Each peptide contained a 9-arginine cell transducing sequence (Reference20) positioned at the COOH-terminal and an 11-18 amino acid sequencefrom the vaccinia virus A52R protein. Three peptides (P5, P6, and P14)were found to be insoluble and were eliminated from evaluation. Theremaining 15 peptides were evaluated for their effect on cell viabilityby trypan blue exclusion staining over a range of concentrations andthen each peptide was tested for cytokine inhibition at the maximumconcentration that had no effect on cell viability. Using theFITC-labeled peptides, each of the peptides was shown to be internalizedinto RAW264.7 cells, a mouse monocyte/macrophage cell line, as assessedby FACS. The necessity of the cell transducing sequence for cellularinternalization was demonstrated when RAW264.7 cells were incubated withone of the FITC-labeled peptides (peptide P13) and internalizationassessed by FACS. The amount of FITC-peptide that was internalized intocells produced a geometric mean fluorescent unit (F) value of 151 (FIG.1). As a control, peptide P13 was produced that contained the 11 aminoacid sequence from A52R but lacked the 9-arginine transduction sequence.This FITC-labeled control peptide showed significantly lessinternalization (F=17) into RAW264.7 cells than the peptide containingthe transduction sequence and was similar to the background level seenwhen cells were incubated with medium without peptide (F=8). In initialexperiments, individual peptides (lacking the FITC-label) were examinedfor inhibition of MIP-2 secretion from RAW264.7 cells. MIP-2 (macrophageinflammatory protein-2), a neutrophil chemoattractant factor, isimportant in development of inflammation. As a control, each peptide wastested for its effect on cytokine secretion without any addedstimulants. These studies demonstrated that all peptides caused <4 ng/mlof MIP-2 secretion in the absence of a stimulus. Individual peptideswere then examined for inhibition of MIP-2 secretion from RAW264.7 cellsactivated by a variety of PAMPs (LPS, Poly(I:C), CpG-ODN). Some peptidesdemonstrated moderate inhibition of MIP-2 secretion while the majorityof peptides examined had no significant effect on cytokine secretion, asdemonstrated when cells were stimulated with CpG-ODN (FIG. 2). Onepeptide (P13), with the amino acid sequence DIVKLTVYDCI-RRRRRRRRR (SEQID NO: 20), demonstrated significant inhibition of MIP-2 secretion foreach of the TLR ligands examined and was used for furthercharacterization. A scrambled peptide of P13 (ITCVDVDLIYK-RRRRRRRRR—SEQID NO: 21) was also produced as a negative control.

Inhibition of cytokine secretion: The initial studies with peptide P13examined its effect on MIP-2 secretion at one concentration (10 μM) inresponse to the stimulants LPS, CpG ODN, and Poly(I:C). Cell viabilitystudies confirmed that a 10 μM concentration of peptides P13 and thescrambled P13 control had no effect on cell viability. Peptide P13 wasthen examined at various concentrations for inhibition of cytokinesecretion in response to these and other TLR ligands. RAW264.7 cellswere incubated for 15 minutes with 5, 8, or 10 μM of peptide P13 andthen stimulated with either CpG-ODN, LPS, Poly(I:C), flagellin, orzymosan for 18 hours. Cell-free supernatants from treated cells wereassessed for MIP-2 by ELISA. Treatment with peptide significantlyinhibited MIP-2 secretion for each of the 5 TLR ligands examined (FIGS.3-7). Peptide inhibition of MIP-2 secretion was dose-dependent for theTLR ligands LPS, Poly(I:C), and flagellin. Inhibition was most dramaticwhen cells were stimulated with CpG ODN, and ranged from approximately90% to 35%, depending upon the TLR ligand used for cell activation.Testing of the control scrambled peptide at 10 mM, under identicalexperimental conditions, showed no inhibition of MIP-2 secretion inresponse to the five PAMPs examined above. To determine if peptide P13would be effective in inhibiting cytokine secretion induced by acombination of stimuli, RAW264.7 cells were incubated with both LPS (0.5ng/ml) and CpG-ODN (0.5 ug/ml). Each stimulus was used at half of itsoptimal stimulatory concentration. Incubation with 10 μM peptide P13reduced MIP-2 secretion 81%. We next sought to establish the effect onMIP-2 secretion when peptide was added at various time points before,simultaneous with, or after stimulation with CpG-ODN. Inhibition seenafter addition of peptide P13 up to one hour after stimulation withCpG-ODN was similar to inhibition seen when peptide was added eitherbefore or simultaneous with CpG-ODN (>85% inhibition). Significantinhibition of MIP-2 secretion was demonstrated even when peptide wasadded as long as 4 hours after stimulation of cells with CpG-ODN (FIG.8). Peptide inhibition of cytokines other than MIP-2 was also examined.RAW264.7 cells were stimulated with CpG-ODN and secretion of TNF-α,IL-6, and IL-10 quantified by ELISA. Treatment of cells with peptide P13significantly inhibited secretion of each of these cytokines (FIGS.9-11). In addition, peptide inhibited intracellular TNF-α production by59% as assessed by FACS. Inhibition of cytokine secretion by peptide P13was also seen when human BJAB B cells were activated by TLR ligands. Insummary, the peptide demonstrated inhibition of cytokines stimulated bynumerous TLR ligands, both alone and in combination. The inhibition wasdose-dependent and seen for a variety of cytokines produced by bothmacrophages and B cells. Peptide P13 was effective even when added afterthe stimulating PAMP, suggesting a potential application as ananti-inflammatory therapy.

Mechanism of peptide P13 inhibition of cytokine secretion: The A52Rprotein has been previously demonstrated to inhibit TIR signaling byinteracting with both IRAK2 (interleukin-1 receptor-associated kinase 2)and TRAF6 (TNF (tumor necrosis factor) receptor associated factor 6),intracellular signaling molecules involved in TIR signaling (Reference18). We hypothesize that P13, like the parent protein, inhibits cytokinesecretion through interaction with IRAK2 and/or TRAF6. Data from thefollowing experiments support this hypothesis. i) Peptide P13 must beinternalized to inhibit cytokine secretion. To interact with IRAK2and/or TRAF6, peptide P13 must be internalized. We compared treatment ofcells with peptide P13 that either contained or lacked the 9-argininecell transducing sequence. Peptide without the transducing sequence wasnot internalized into cells as previously demonstrated (FIG. 1).Treatment of RAW264.7 cells with peptide P13 lacking the transducingsequence had no effect on MIP-2 secretion in response to stimulationwith either LPS or CpG-ODN (Table I). As previously demonstrated,peptide containing the cell transducing sequence significantly inhibitedMIP-2 secretion (FIGS. 3-7). ii) Peptide P13 does not inhibit cytokinesecretion stimulated by PMA or TNF-α. Both PMA and TNF-α activateRAW264.7 cells via signaling pathways independent of either IRAK2 orTRAF6, resulting in secretion of MIP-2. Treatment with peptide had noaffect on MIP-2 secretion in response to stimulation with either PMA orTNF-α (Table II). iii) Peptide P13 inhibits phosphorylation of IκB-α.The intracellular signaling pathway triggered by the interaction ofPAMPs with TLRs involves the IRAK (interleukin-1 receptor-associatedkinase) family and TRAF6, resulting in translocation of NF-κB to thenucleus, followed by secretion of pro-inflammatory cytokines. Activationof NF-κB is dependent on the phosphorylation and proteolysis of the IκBproteins. RAW264.7 cells were treated with either peptide P13 or controlscrambled peptide and stimulated with LPS for either 15 or 30 minutes.Cells were lysed and analyzed by immunblotting using Phospho-IκB-αantibody, which detects endogenous levels of IκB-α only whenphosphorylated at Ser32. Peptide P13 completely inhibited thephosphorylation of IκB-α in LPS activated cells as compared to cellstreated with control scrambled peptide, which demonstrated a twofoldincrease overbackground (Table III). iv) Peptide P13 inhibits cytokinesecretion initiated by TLR3. The TLR3 signaling pathway is differentfrom the other TLR signaling pathways in that it requires TRAF6, but notthe IRAK family or the upstream adaptor molecule MyD88, for theproduction of pro-inflammatory cytokines. Downstream of TRAF6, thepathways are similar, both resulting in the phosphorylation of IκB andthe translocation of NF-κB to the nucleus. As demonstrated above,peptide P13 inhibits MIP-2 production from RAW264.7 cells stimulatedwith Poly(I:C), a synthetic ligand for TLR3 (FIG. 5). Collectively,these data support the conclusion that peptide P13 inhibits cytokinesecretion by interaction with an intracellular portion of the TIRsignaling pathway upstream of IκB. The inhibition of TLR3 mediatedcytokine secretion, in combination with the other PAMP/TLR inhibitorydata, suggests that the effect of peptide P13 is on TRAF6 or adownstream component of the TIR signaling pathway. The data areconsistent with the hypothesis that peptide P13, like the parent A52Rprotein, interacts in the TIR signaling pathway at TRAF6.

Inhibition of middle ear inflammation: The effect of peptide P13 onbacterial-induced inflammation in vivo was examined using a murine modelof otitis media with effusion (OME). The inflammatory response inbacterial-induced OME is initiated by TLR activation and ischaracterized by infiltration of cells into the middle ear, fluidaccumulation, and thickening of the mucosal epithelium and the tympanicmembrane (Reference 21). To first examine any potential effects causedby peptide alone without added bacteria, five mice were injected in oneear with PBS and in the opposite ear with 10 μM peptide P13. Three dayslater the animals were killed, middle ears embedded, sectioned, stainedand evaluated for fluid area, infiltrating cell number, and thickness ofthe tympanic membrane. Paired t-tests (2-tailed) were used to analyzeeach of the three parameters. In the absence of bacterial-inducedinflammation, no differences were seen between the PBS injected ear andpeptide P13 injected ear in i) fluid area (p=0.104), ii) cell number(p=0.880), or iii) tympanic membrane thickness (p=0.891). To examine theeffectiveness of the peptide to affect inflammation in vivo, twentyBALB/c mice were injected in the middle ear on one side withheat-inactivated S. pneumoniae plus PBS and in the middle ear on theopposite side with heat-inactivated S. pneumoniae plus 10 μM peptideP13. Three days later the animals were killed, and evaluated for middleear fluid area, infiltrating cell number, and thickness of the tympanicmembrane. Disease development was defined as an increase over backgroundcontrols (PBS injected ears n=18) of at least two standard deviations intwo out of the three parameters quantified. A total of 7 out of 20 micemet the criteria for disease induction. Analysis of middle ears bypaired t-tests from these 7 mice with disease showed that peptidetreatment significantly reduced the amount of fluid (p=0.004),infiltrating cell number (p=0.02), and thickness of the tympanicmembrane (p=0.002), all parameters of middle ear inflammation (TableIV). Examination of these three parameters of inflammation for eachindividual mouse with disease illustrates the dramatic effect seen witha single treatment of peptide P13 (Table V). Of interest, 6 out of the 7mice demonstrated reductions in all areas of inflammation, while oneanimal (#4-182) showed only modest reduction in fluid area and tympanicmembrane thickness, and no reduction in cell number. Photographs from anormal, non-diseased animal and a representative animal with diseaseillustrate the effect of peptide on bacterial-induced inflammation invivo. The middle ear of normal mice is free of fluid or cells (FIG. 12)and the mucosal epithelium that lines the middle ear space is normallycomprised of 1-2 low cuboidal cells (FIG. 13). Injection of heat-killedbacteria resulted in a marked inflammatory response in the middle earafter 3 days. This was characterized by mucosal and tympanic membraneswelling, cellular infiltration, and significant fluid (effusion)secretion and accumulation that filled the middle ear space (FIGS. 14and 15). The inflammatory response led to significant mucosal cellularhypertrophy and active secretion of mucins and other fluids (FIG. 16).When peptide P13 was injected with the bacteria, a significant reductionwas seen in fluid accumulation into the middle ear space (FIG. 17) andreduced mucosal hypertrophy (FIG. 18).

Inhibition of inflammatory mediators in a murine septic shock model:Preliminary data has been collected documenting the inhibitory effect ofpeptide P13 on inflammatory mediators in a murine septic shock model.BALB/c mice (4 animals/group) were injected i.p. with PBS, LPS at 100μg/mouse/250 μl, or 100 μg LPS plus various doses of peptide P13. Serumwas collected at 2 and 6 hours after treatment and evaluated for thepro-inflammatory cytokines MIP-2 and TNF-α by ELISA, and for soluableICAM-1. The animals injected simultaneously with peptide P13 and 100 μgLPS showed up to a 31% reduction in MIP-2, a 60% reduction in TNF-α, anda 35% reduction in soluable ICAM-1 as compared to animals injected onlywith LPS.

Other immunomodulatory peptides derived from A52R: Eighteen peptideswere designed and constructed based on the sequence of the vacciniavirus A52R protein as described previously in this manuscript. Eachpeptide contained a nine-arginine cell transducing sequence positionedat the C terminus and an 11- to 18-aa sequence from the vaccinia virusA52R protein (FIG. 19). Three peptides (P5, P6, and P14) were found tobe insoluble and were eliminated from evaluation. Each of the remaining15 peptides were examined for inhibition of MIP-2 secretion fromRAW264.7 cells activated by the PAMPs LPS, poly(I:C) and CpG-ODN.Peptide P13 was found to have the greatest inhibitory activity and wasused for further characterization as previously described. Several otherpeptides were also identified for further study, based either on thepeptide's inhibitory activity, or on the ability of the peptide toenhance cytokine production. Using the data generated by stimulationwith CpG-ODN, P13 was selected for it's ability to inhibit cytokineactivity, and P10 for it's cytokine enhancing ability (FIG. 2). Cellularstimulation with LPS (FIG. 20) yielded three peptides that significantlyenhanced MIP-2 production, (P2, P4, P9), and three that inhibitedcytokine activity (P7, P13, P16). Data from cell stimulated withpoly(I:C) showed four peptides of interest, P3, which increases MIP-2production, and P13, P16 and P18, which show inhibitory activity (FIG.21). In summary, we have demonstrated that peptide P13 (FIG. 22) is apotent inhibitor of cytokine secretion and bacterial-inducedinflammation. In addition to peptide P13, we demonstrated that peptidesP7, P16 and P18 (FIG. 22) also inhibited cytokine activity. Peptides P2,P3, P4, P9 and P10 (FIG. 23) demonstrated enhanced cytokine activity.

TABLE I Peptide P13 Lacking a Cell Transducing Sequence Fails to InhibitMIP-2 Secretion MIP-2 Treatment (pg/ml +/− S.D.)^(a) % Inhibition LPS46,618 +/− 923 LPS + peptide P13 12,435 +/− 269 73% LPS + peptide P13(no  46,931 +/− 1335  0% transducing sequence) CpG-ODN 31,194 +/− 743CpG-ODN + peptide P13   3242 +/− 238 90% CpG-ODN + peptide P13 29,312+/− 618  6% (no transducing sequence) ^(a)RAW264.7 cells were incubated15 minutes with either medium, peptide P13 containing the transducingsequence, or peptide P13 lacking the transducing sequence and thenstimulated with either LPS (1 ng/ml) or CpG ODN (1 μg/ml). Cell-freesupernatants were analyzed for MIP-2 by ELISA and data expressed aspg/ml +/− S.D.

TABLE II Peptide P13 Does Not Inhibit Non-TLR Induced MIP-2 SecretionMIP-2 pg/ml Treatment ^(a) (+/−S.D.) Medium 781 +/− 7 TNF-α 1744 +/− 16TNF-α + peptide P13 2384 +/− 16 PMA 22,144 +/− 544  PMA + peptide P13 24,736 +/− 1216 ^(a) RAW264.7 cells were incubated 15 minutes witheither medium or peptide P13 and then stimulated with either medium,TNF-α (100 ng/ml) or PMA (100 ng/ml) for 18 hours. Cell-freesupernatants were analyzed for MIP-2 by ELISA and data expressed aspg/ml +/− S.D.

TABLE III Peptide P13 Inhibits Phosphorylation of IκB-α PhosphorylatedIκB-α Band Intensity/Area^(a) Scrambled Peptide Treatment peptide P13medium 6.3 5.4 LPS (15 min) 13.1 4.9 LPS (30 min) 13.3 3.1 ^(a)RAW264.7cells were incubated 15 minutes with either peptide P13 or controlscrambled peptide and then treated with either medium, or LPS (1 ng/ml)for either 15 or 30 minutes. Immunoblotting was performed usingphospo-IκB-α (ser32) antibody. Measurements of band intensity were madeusing the Nucleo Tech Gel Expert Software linked to an Epson expression636 scanner and data expressed as band intensity/area.

TABLE IV Peptide P13 Inhibition of Middle Ear Inflammation^(a) TympanicMembrane Fluid Area Cell Number Thickness Treatment (microns² +/− S.D.)(+/−S.D.) (microns +/− S.D.) PBS^(b) 1016 +/− 1397 31 +/− 41 44 +/− 20S. pneumoniae ^(c) 5771 +/− 2077 252 +/− 140 105 +/− 33  S. pneumoniae +1486 +/− 1192 111 +/− 119 44 +/− 15 peptide P13^(c) p value(2-tailed)^(d) p = 0.004 p = 0.020 p = 0.002 ^(a)Middle ear inflammationwas assessed by measuring three consecutive tissue sections for area offluid in the middle ear, number of cells in the middle ear fluid, andthickness of the tympanic membrane measured at a point away from theinjection site. Data represent the mean +/− SD of 7 animals with middleear inflammation. Statistical evaluation was done using a paired t-test.^(b)The PBS treated animals (n = 18) received no bacteria or peptideP13. ^(c)Animals (n = 7) injected in one ear with S. pneumoniae plus PBSand in the opposite ear injected with S. pneumoniae plus peptide P13 (10μM). ^(d)Statistical evaluation using a paired t-test was done usingdata collected from diseased animals (n = 7) injected with bacteria andcomparing peptide vs. no peptide P13 treatment.

TABLE V Peptide P13 Inhibits Development of Fluid, Cell Number, andTympanic Membrane Thickening in a Murine Model of OME ^(a) Tympanicmembrane Fluid Area Cell Number thickness Animal % inhibition %inhibition % inhibition #4-21 84% 39% 63% #4-24 85% 96% 65% #4-177 86%77% 66% #4-182 11% 0 22% #4-183 73% 44% 71% #4-185 66% 77% 60% #4-19594% 89% 69% ^(a) Middle ear inflammation was assessed as described inTable III. Percent inhibition is calculated by comparing fluid area,cell number, and tympanic membrane thickness seen in one ear injectedwith S. pneumoniae plus PBS with the same parameters of inflammationseen in the opposite ear injected with S. pneumoniae plus peptide P13(10 μM).

While this invention has been described as having preferred sequences,ranges, steps, materials, structures, features, and/or designs, it isunderstood that it is capable of further modifications, uses and/oradaptations of the invention following in general the principle of theinvention, and including such departures from the present disclosure asthose come within the known or customary practice in the art to whichthe invention pertains, and as may be applied to the central featureshereinbefore set forth, and fall within the scope of the invention andof the limits of the appended claims.

REFERENCES

The following references, and those cited in the disclosure herein, arehereby incorporated herein in their entirety by reference.

-   1. Takeda, K., and S. Akira. 2004. TLR signaling pathways. Seminars    in Immunology 16:3.-   2. Schnare M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, and R.    Medzhitov. 2001. Toll-like receptor control activation of adaptive    immune responses. Nat. Immuno. 2:947.-   3. Granucci, F., C. Vizzardelli, N. Pavelka, S. Feau, M. Persico, E.    Virzi, M. Rescigno, G. Moro, and P. Ricciardi-Castagnoli. 2001.    Inducible 11-2 production by dendritic cells revealed by global gene    expression analysis. Nat. Immunol. 2:882.-   4. Krieg, A. M. 2002. CpG motifs in bacterial DNA and their immune    effects. Ann. Rev. Immunol. 20:709.

5. Trinchieri, G. 1998. Interleukin-12: a cytokine at the interface ofinflammation and immunity. Adv. Immunol. 70:83.

-   6. Ozato, K., H. Tsujimura, and T Tamura. 2002. Toll-like receptor    signaling and regulation of cytokine gene expression in the immune    system. BioTechniques Oct. Suppl:66.-   7. Yi, A. K., J. G. Yoon, S. J. Yeo, S.C. Hong, B. K. English,    and A. M. Krieg. 2002. Role of mitogen-activated protein kinases in    CpG DNA-mediated IL-10 and IL-112 production: central role of    extracellular signal-regulated kinase in the negative feedback loop    of the CpG DNA-mediated Th1 response. J. Immunol. 168:4711.-   8. Fan, J. and A. B. Malik. 2003. Toll-like receptor-4(TLR4)    signaling augments chemokine-induced neutrophil migration by    modulating cell surface expression of chemokine receptors. Nat. Med.    9:315.-   9. McCoy, S. L., S. E. Kurtz, F. A. Hausman, S. R. Trune, R. M.    Bennett, and S. H. Hefeneider. 2004. Activation of RAW264.7    macrophages by bacterial DNA and lipopolysaccharide increases cell    surface DNA binding and internalization. J. Biol. Chem. 279:17217.-   10. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y.    Takeda, K. Takeda, and S. Akira. 1999. Cutting Edge: Toll-like    receptor 4 (TLR4)-deficient mice are hyproresponsive to    lipopolysaccharide: evidence for TLR4 as the Lps gene product. J.    Immunol. 162:3749.-   11. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H.    Sanjo, M. Matsumo, K. Hoshino, H. Wagner, K. Takeda, and S.    Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature    408:740.-   12. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C.    Yi, D. R. Goodlett, J. K. Eng, S. Akira, D, M. Underhill, and A.    Aderem. 2001. The innate immune response to bacterial flagellin is    mediated by Toll-like receptor 5. Nature 410:1099.-   13. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors.    Ann. Rev. Immunol. 21:335.

14. Akira, S. 2003. Mammalian Toll-like receptors. Curr. Opin. Immunol.15:5.

-   15. Bowie, A., E. Kiss-Toth, J. A. Symons, G. L. Smith, S. K. Dower,    and L. A. J. O'Neill. 2000. A46R and A52R from vaccinia virus are    antagonists of host IL-1 and toll-like receptor signaling. Proc.    Natl. Acad. Sci. U.S.A. 97:10162.-   16. O'Neill L. 2000. The Toll/interleukin-1 receptor domain: a    molecular switch for inflammation and host defence. Biochem. Soc.    Trans. 28:557.-   17. Bellows, C. F., R. F. Garry, and B. M. Jaffe. 2003. Vaccinia    virus-induced inhibition of nitric oxide production. J. Surg. Res.    111:127.-   18. Harte, M. T., I. R. Haga, G. Maloney, P. Gray, P. C.    Reading, N. W. Bartlett, G. L. Smith, A. Bowie, and L. A. J.    O'Neill. 2003. The poxvirus protein A52R targets Toll-like receptor    signaling complexes to suppress host defense. J. Exp. Med. 197:343.-   19. Yi, A. K., and A. M. Krieg. 1998. Cutting Edge: Rapid induction    of mitogen-activated protein kinases by immune stimulatory CpG    DNA. J. Immunol. 161:4493.-   20. Wender, P. A., D. J. Mitchell, K. Pattabiraman, E. T. Pelkey,    and L. Steinman. 2000. The design, synthesis, and evaluation of    molecules that enable or enhance cellular uptake: Peptoid molecular    transporters. Proc. Natl. Acad. Sci. U.S.A. 97:13003.-   21. Barzilai A., B. Dekel, R. Dagan, and E. Leibovitz. 2000. Middle    ear effusion 11-6 concentration in bacterial and non-bacterial acute    otitis media. Acta Paediatr 89:1068.-   22. Takeda, K. and S. Akira. 2004. TLR signaling pathways. Semin.    Immunol. 16:3.-   23. Janssens, S., and R. Beyaert. 2003. Functional diversity and    regulation of different interleukin-1 receptor-associated kinase    (IRAK) family members. Mol. Cell. 11:293.-   24. Daun, J. M., and M. J. Fenton. 2000. Interleukin-1/Toll receptor    family members: receptor structure and signal transduction    pathways. J. Interferon Cytokine Res. 20:843.-   25. Barton, G. M., and R. Medzhitov. 2003. Linking Toll-like    receptors to IFN-α/β expression. Nat. Immunol. 4:432.-   26. Karasen R. M., Y. Sutbeyaz, B. Aktan, H. Ozdemir, and C.    Gundogu. 2000. Effect of web 2170 BS, platelet activating factor    receptor inhibitor, in the guinea pig model of middle ear    inflammation. Ann Otol Rhinol Laryngol 109:549.-   27. Daly, K. A., L. L. Hunter, and G. S. Giebink. 1999. Chronic    Otitis Media with Effusion. Pediatrics in Review 20:85.-   28. Kubba H., J. P. Pearson, and J. P. Birchall. 2000. The aetiology    of otitis media with effusion: a review. Clin Otolaryngol 25:181.-   29. O'Neill, L. A. J. 2003. Therapeutic targeting of Toll-like    receptors for inflammatory and infectious diseases. Curr. Opin.    Pharm. 3:396.-   30. Zuany-Amorim, C., J. Hastewell, and C. Walker. 2002. Toll-like    receptors as potential therapeutic targets for multiple diseases.    Nat. Rev. Drug Discov. 1:797.-   31. Ikezoe, T., Y. Yang, D. Heber, H. Taguchi, and H. P.    Koeffler. 2003. PC-SPES: A potent inhibitor of nuclear factor-κB    rescues mice from lipopolysaccharide-induced septic shock. Mol.    Pharmacol. 64:1521.-   32. Delgado, M., C. Abad, C. Martinez, M. G. Juarranz, J. Leceta, D.    Ganea, and R. P. Gomariz. 2003. PACAP in immunity and inflammation.    Ann. N.Y. Acad. Sci. 992:141.-   33. Basu, S., and M. J. Fenton. 2004. Toll-like receptors: function    and roles in lung disease. Am. J. Physiol. Lung Cell Mol. Physiol.    286:L887.-   34. Kopp, E., and S. Ghosh. 1994. Inhibition of NF-kappa B by sodium    salicylate and aspirin. Science 265:956.-   35. Almawi, W. Y., and O. K. Melemedjian. 2002. Negative regulation    of nuclear factor-kappaB activation and function by    glucocorticoids. J. Mol. Endocrinol. 28:69.-   36. Andreakos, E. T., B. M. Foxwell, F. M. Brennan, R. N. Maini,    and M. Feldmann. 2002. Cytokines and anti-cytokine biologicals in    autoimmunity: present and future. Cytokine Growth Factor Rev.    13:299.-   37. Meng, G., M. Rutz, M. Schiemann, J. Metzger, A. Grabiec, R.    Schwandner, P. B. Luppa, F. Ebel, D. H. Busch, S. Bauer, H. Wagner,    and C. J. Kirschning. 2004. Antagonistic antibody prevents Toll-like    receptor 2-driven lethal shock-like syndromes. J. Clin. Invest.    113:1473.-   38. Sweet, M. J., B. P. Leung, D. Kang, M. Sogaard, K. Schulz, V.    Trajkovic, C. C. Campbell, D. Xu, and F. Y. Liew. 2001. A novel    pathway regulating lipopolysaccharide-induced shock by ST2/T1 via    inhibition of Toll-like receptor 4 expression. J. Immunol. 166:6633.-   39. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. McKenzie, L. A.    O'Neill, and F. Y. Liew. 2004. ST2 is an inhibitor of interleukin 1    receptor and Toll-like receptor 4 signaling and maintains endotoxin    tolerance. Nat. Immunol. 5:373.-   40. Chuang, T. H., and R. J. Ulevitch. 2004. Triad3A, an E3    ubiquitin-protein ligase regulating Toll-like receptors. Nat.    Immunol. 5:495.-   41. Bartfai, T., M. M. Behrens, S. Gaidarova, J. Pemberton, A.    Shivanyuk, and J. Rebek, Jr. 2003. A low molecular weight mimic of    the Toll/IL-1 receptor/resistance domain inhibits IL-1    receptor-mediated responses. Proc. Natl. Acad. Sci. U.S.A. 100:7971.-   42. McCoy, S. L., Kurtz, S. E., MacArthur, C. J., Trune, D. R, and    Hefeneider, S. H. 2005. Identification of a Peptide Derived from    Vaccinia Virus A52R Protein That Inhibits Cytokine Secretion in    Response to TLR-Dependent Signaling and Reduces In Vivo    Bacterial-Induced Inflammation. Journal of Immunology, 174:    3006-3014.

1. An isolated polypeptide consisting of one of (a) the amino acidsequence set forth as SEQ ID NO: 7, (b) a fusion protein comprising theamino acid sequence set forth as SEQ ID NO: 7 and a heterologousprotein; (c) the amino acid sequence set forth as SEQ ID NO: 7 and atransducing sequence.
 2. The isolated polypeptide of claim 1, whereinthe transducing sequence is nine arginine residues.
 3. The isolatepolypeptide of claim 1, wherein the heterologous protein is a markerprotein.
 4. The isolated polypeptide of claim 1, consisting of the aminoacid sequence set forth as SEQ ID NO:
 7. 5. The isolated polypeptide ofclaim 1, consisting of the amino acid sequence set forth as SEQ ID NO: 7and a transducing sequence.
 6. A composition comprising an effectiveamount of the polypeptide of claim 1 and a pharmaceutical carrier.
 7. Amethod of inhibiting Toll Like Receptor (TLR)-induced cytokine secretioncomprising: administering to a subject in need thereof, an effectiveamount of the isolated polypeptide of claim 1, thereby inhibiting TollLike Receptor (TLR)-induced cytokine secretion.
 8. The method of claim7, comprising administering to the subject an effective amount of apeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7and a transducing sequence; thereby inhibiting Toll Like Receptor(TLR)-induced cytokine secretion.
 9. The method of claim 7, wherein thepeptide consists of SEQ ID NO: 7 and nine consecutive arginine residues.10. A method of reducing or inhibiting Toll Like Receptor (TLR)-inducedinflammation, comprising: administering to a subject in need thereof, aneffective amount of one of: a) a peptide consisting of the amino acidsequence set forth in SEQ ID NO: 7; or b) an effective amount of apeptide consisting of the amino acid sequence set forth in SEQ ID NO:7and a transducing sequence; thereby reducing or inhibiting Toll LikeReceptor (TLR)-induced inflammation.
 11. The method of claim 10, whereinan effective amount of a peptide consisting of the amino acid sequenceset forth in SEQ ID NO: 7 and a transducing sequence is administered.12. The method of claim 10, wherein the polypeptide consists of SEQ IDNO:
 7. 13. The method of claim 10, wherein the inflammation is caused bya bacteria.
 14. The method of claim 10, wherein the bacteria isStreptococcus pneumoniae.
 15. The method of claim 10, wherein thesubject has otitis media.
 16. The method of claim 10, wherein: TLR isone or more of TLR 2, TLR3, TLR4, TLR5 and TLR9.
 17. The method of claim10, wherein TLR is TLR2 or TLR3.
 18. The method of claim 10, wherein TLRis TLR4, TLR5 or TLR9.